Method for producing factor G derived from horseshoe crab

ABSTRACT

The invention provides a virus harboring a DNA encoding a subunit of limulus-derived factor G, the virus being capable of mass-producing a (1→3)-β-D-glucan assay reagent of satisfactory quality, steadily and at low cost, a cell harboring the virus, and a method of producing factor G by use of the cell.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/665,779, filed on Mar. 28, 2005.

FIELD OF THE INVENTION

The present invention relates to a virus harboring DNA encoding asubunit of factor G derived from a horseshoe crab (hereinafter may bereferred to as limulus-derived factor G), to a cell harboring the virus,and to a method of producing factor G by use of the cell.

BACKGROUND ART

Abbreviations used in the present specification are as follows.

AcNPV: nuclear polyhedrosis virus of Autographa californica

BG: (1→3)-β-D-glucan

Et: endotoxin (also referred to as lipopolysaccharide)

HEPES: 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid

HRP: horseradish peroxidase

MOI: multiplicity of infection

NPV: nuclear polyhedrosis virus

PBS: phosphate buffered saline

PCR: polymerase chain reaction

pNA: p-nitroaniline

PVDF: polyvinylidene difluoride

SDS: sodium dodecyl sulfate

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Japanese Patent Application laid-Open (kokai) No. 08-122334 and anon-patent document (J. Protein Chem., 5, p. 255-268 (1986)) disclosemethods for determining Et or BG by use of an amebocyte lysate of ahorseshoe crab (hereinafter referred to simply as a lysate). Thesemethods are based on coagulation of the lysate by Et or BG. Thecoagulation reaction occurs through cascade reaction of coagulationfactors.

For example, when BG is brought into contact with the lysate, factor Gcontained in the lysate is activated, to thereby form activated factorG. The activated factor G activates a pro-clotting enzyme present in thelysate, to thereby form a clotting enzyme. The clotting enzymehydrolyzes a specific site of a coagulogen molecule present in thelysate, thereby forming coagulin gel, leading to coagulation of thelysate. The coagulogen also acts on a synthetic substrate (e.g.,t-butoxycarbonyl-leucyl-glycyl-arginine-pNA (Boc-Leu-Gly-Arg-pNA)), tothereby hydrolyze the amide bonds, whereby pNA is released. Thus, BG canbe determined through measuring absorbance of the formed coloringsubstance (pNA) (disclosed in Japanese Patent Application laid-Open(kokai) No. 08-122334).

Factor G is a protein formed of subunits α and β, and cloning of eachsubunit has already been performed (disclosed in J. Biol. Chem., 269(2),p. 1370-1374 (1994)). However, an active protein (factor G) has beendifficult to express through employment of cloned DNAs encoding thesubunits.

SUMMARY OF THE INVENTION

Thus, an object of the present invention is provide an virus harboring aDNA encoding a subunit of limulus-derived factor G, the virus beingcapable of mass-producing a BG assay reagent of satisfactory quality,steadily and at low cost. Another object is to provide a cell harboringthe virus. Still another object is to provide a method of producingfactor G by use of the cell.

The present inventors have conducted extensive studies in order toattain the aforementioned objects, and have found that a protein havingfactor G activity can be produced by use of a cell harboring a viruscontaining a DNA encoding a subunit of factor G, whereby a BG assayreagent of satisfactory quality can be mass-produced steadily and at lowcost. The present invention has been accomplished on the basis of thisfinding.

Accordingly, the present invention provides a virus harboring a DNAencoding subunit α of limulus-derived factor G (hereinafter the virusmay be referred to as “virus 1 of the present invention”). The horseshoecrab (limulus) is preferably selected from among Tachypleus tridentatus,Limulus polyphemus, Tachypleus gigas, and Carcinoscorpius rotundicauda.

The DNA encoding subunit α of limulus-derived factor G is preferably aDNA (A) or a DNA (B) as described below:

(A) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 2,

(B) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 2 in which one or more amino acid residues are deleted,substituted, inserted, or transposed and having activity of subunit α oflimulus-derived factor G.

The DNA encoding subunit α of limulus-derived factor G herein is alsopreferably a DNA (a) or a DNA (b) as described below:

(a) a DNA having a nucleotide sequence defined by nucleotides 1 to 2022in SEQ ID NO: 1,

(b) a DNA having a nucleotide mutation in a nucleotide sequence definedby nucleotides 1 to 2022 in SEQ ID NO: 1, the mutation causing deletion,substitution, insertion, or transposition of one or more amino acidresidues in the amino acid sequence of a protein encoded by themutation-containing nucleotide sequence, and the expressed proteinhaving activity of subunit α of limulus-derived factor G.

The virus is preferably baculovirus. The baculovirus is preferably NPV.The NPV is preferably AcNPV.

The present invention also provides a virus harboring a DNA encodingsubunit β of limulus-derived factor G (hereinafter the virus may bereferred to as “virus 2 of the present invention”).

The horseshoe crab (limulus) is preferably selected from amongTachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, andCarcinoscorpius rotundicauda.

The DNA encoding subunit β of limulus-derived factor G herein is alsopreferably a DNA (A) or a DNA (B) as described below:

(A) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 4,

(B) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 4 in which one or more amino acid residues are deleted,substituted, inserted, or transposed and having activity of subunit β oflimulus-derived factor G.

The DNA encoding subunit β of limulus-derived factor G herein is alsopreferably a DNA (a) or a DNA (b) as described below:

(a) a DNA having a nucleotide sequence defined by nucleotides 1 to 930in SEQ ID NO: 3,

(b) a DNA having a nucleotide mutation in a nucleotide sequence definedby nucleotides 1 to 930 in SEQ ID NO: 3, the mutation causing deletion,substitution, insertion, or transposition of one or more amino acidresidues in the amino acid sequence of a protein encoded by themutation-containing nucleotide sequence, and the expressed proteinhaving activity of subunit β of limulus-derived factor G.

The virus is preferably baculovirus. The baculovirus is preferably NPV.The NPV is preferably AcNPV.

Hereinafter, virus 1 of the present invention and virus 2 of the presentinvention may be collectively or individually referred to as “the virusof the present invention”.

The present invention also provides a cell harboring the virus of thepresent invention (hereinafter the cell may be referred to as “the cellof the present invention”).

The cell of the present invention preferably harbors viruses 1 and 2 ofthe present invention. Preferably, the cell is obtained throughinfection with virus 1 and virus 2 such that MOI of virus 1 exceeds MOIof virus 2. In this case, the ratio of MOI of virus 1 to MOI of virus 2is preferably controlled to 1.5:1 to 64:1.

The cell of the present invention is preferably a cell of insect origin.

The present invention also provides a method of producing subunit αand/or subunit β of limulus-derived factor G, the method comprisinggrowing the cell of the present invention and collecting subunit αand/or subunit β of limulus-derived factor G from the growth product(hereinafter the method may be referred to as “the method of the presentinvention”). The subunit α and/or subunit β of limulus-derived factor Gis preferably a protein which is formed of subunit α and subunit β andwhich maintains limulus-derived factor G activity.

The method of the present invention includes a concept of “a method ofproducing factor G, the method comprising growing a cell which harbors aDNA encoding subunit α of factor G derived from a horseshoe crab and aDNA encoding subunit β of factor G derived from a horseshoe crab, andcollecting, from the growth product, a protein having activity of factorG derived from a horseshoe crab”.

The virus of the present invention is very useful, since the cell of thepresent invention, which is useful for mass-producing factor G ofsatisfactory quality, steadily, at high efficiency and low cost, can beattained by using the virus.

Employment of the cell is remarkably useful, since a protein whichmaintains factor G activity and which has satisfactory quality can bemass-produced steadily at high efficiency and low cost, whereby themethod of the present invention can be attained. Furthermore, throughemployment of the method of the present invention, a protein whichmaintains factor G activity and which has satisfactory quality can bemass-produced steadily, at high efficiency and low cost.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Best modes for carrying out the present invention will next be describedin detail.

<1>-1 Virus 1 of the Present Invention

Virus 1 of the present invention is a virus harboring a DNA encodingsubunit α of limulus-derived factor G.

No particular limitation is imposed on the type of the DNA encodingsubunit α of limulus-derived factor G harbored by virus 1 of the presentinvention, so long as the DNA encodes subunit α of limulus-derivedfactor G.

Examples of the DNA include those encoding subunit α of factor G derivedfrom the following horseshoe crabs: Tachypleus tridentatus, Limuluspolyphemus, Tachypleus gigas, and Carcinoscorpius rotundicauda.

Of these, DNAs encoding subunit α of factor G derived from Tachypleustridentatus and Limulus polyphemus are preferred, more preferably a DNAencoding subunit α of factor G derived from Tachypleus tridentatus.

Particularly, the DNA harbored by virus 1 of the present invention ispreferably the following DNA (A) or (B):

(A) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 2,

(B) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 2 in which one or more amino acid residues are deleted,substituted, inserted, or transposed and having activity of subunit α oflimulus-derived factor G.

The DNA encoding a protein having an amino acid sequence defined by SEQID NO: 2 herein is a DNA encoding subunit α of factor G derived fromTachypleus tridentatus.

The DNA coding for a naturally occurring protein may includepolymorphism and mutations, and the formed protein may include mutationsin the amino acid sequence due to intracellular alteration ormodification incurred during purification; such as deletion,substitution, insertion, and transposition in amino acid residues.Although having such a mutation, some proteins are known to exhibitphysiological and biological effects virtually the same as those of theprotein having none of the above mutations. Thus, the protein encoded byDNA (B), which slightly differs from the protein encoded by DNA (A) instructure and which has no significant difference in function can beregarded as substantially equivalent to the protein encoded by DNA (A).A similar logic is also applied to the case where the aforementionedmutations are intentionally introduced into an amino acid sequence ofprotein. In this case, a wider range of variants can be fabricated. Forexample, a polypeptide engineered from human interleukin 2 (IL-2) sothat a certain cysteine residue in the amino acid sequence of IL-2 issubstituted by serine is known to maintain human interleukin 2 (IL-2)activity (Science, 224, 1431 (1984)). Also, a certain protein is knownto have a peptide region that is not essential in terms of activity.Examples of such a protein include a signal peptide present in a proteinsecreted from a cell and a pro-sequence observed in a protease precursoror a similar substance. Most of these peptide regions are removed aftertranslation or during conversion to the corresponding activatedproteins. Although having different primary structures, theabove-mentioned variants are virtually equivalent in terms of thefunction to the protein encoded by DNA (A). Therefore, the proteinencoded by DNA (B) represents these proteins.

In the present specification, the term “one or more amino acid residues”refers to amino acid residues which are allowed to have mutationswithout impairing the protein activity. For example, when a proteincontains 600 amino acid residues, the number of such amino acid residuesis about 1 to 30, preferably 1 to 15, more preferably 1 to 8.

The protein encoded by DNA (B) has activity of subunit α oflimulus-derived factor G. Since subunit α of factor G has BG-bindingactivity, subunit α activity can be detected by checking the presence ofBG-binding activity.

The state “harboring a DNA” in virus 1 of the present invention does notexclude the state in which the virus harbors other nucleotides and DNAs,so long as the relevant DNA is harbored. Thus, in addition to the DNA,other DNAs encoding a marker peptide etc. may be harbored.

For example, a vector harboring a linked DNA between the aforementionedDNA (A) or (B) and a DNA encoding a marker peptide etc. also fallswithin the scope of virus 1 of the present invention. When the DNA to beharbored is designed in the above manner, a protein fused with a markerpeptide etc. may be expressed. The thus-expressed protein isadvantageous for facilitating purification, detection, analysis, etc.Examples of the marker peptide include protein A, an insulin signalsequence, His-tag, FLAG, CBP (calmodulin-binding protein), and GST(glutathione S-transferase). For example, a protein fused with protein Amay be purified in a simple manner through affinity chromatographyemploying an IgG-immobilized solid phase. Similarly, a His-tag-fusedprotein may be purified with a magnetic nickel-immobilized solid phase,whereas a FLAG-fused protein may be purified with an anti-FLAGantibody-immobilized solid phase. A protein fused with an insulin signalsequence is secreted from a cell to the outside (e.g., culture medium).Therefore, an extraction step including crushing of cells may beeliminated.

No particular limitation is imposed on the production method of virus 1of the present invention. One exemplary method of producing virus 1 ofthe present invention will be described as follows. More specificprocedure thereof will be described in the Examples.

Firstly, a DNA encoding subunit α of limulus-derived factor G isprovided. In the case where the aforementioned DNA (A) is employed asthe DNA, a DNA encoding a protein having an amino acid sequence definedby SEQ ID NO: 2 is provided. In the case where the aforementioned DNA(B) is employed as the DNA, provided is a DNA encoding a protein havingan amino acid sequence defined by SEQ ID NO: 2 in which one or moreamino acid residues are deleted, substituted, inserted, or transposedand having activity of subunit α of limulus-derived factor G. Noparticular limitation is imposed on the type of the DNA, so long as theDNA encodes the relevant protein. The DNA includes those having avariety of nucleotide sequences due to degeneracy of genetic codes.However, any of these DNAs having a specific nucleotide sequence may beemployed.

The DNA (A) serving as a DNA encoding a protein having an amino acidsequence defined by SEQ ID NO: 2 may be, among others, a DNA having anucleotide sequence defined by nucleotides 1 to 2022 in SEQ ID NO: 1.Alternatively, a DNA deposited in GenBank with an accession No. D16622may also be employed. Furthermore, a DNA having a nucleotide sequencedefined by nucleotides 1 to 2058 in SEQ ID NO: 1 may also be employed.

The DNA (B) serving as a DNA encoding a protein having an amino acidsequence defined by SEQ ID NO: 2 in which one or more amino acidresidues are deleted, substituted, inserted, or transposed and havingactivity of subunit α of limulus-derived factor G, may be theaforementioned DNA (A), a complementary DNA thereof, or a DNA whichhybridizes with any of the DNAs under stringent conditions.

As used herein, the term “stringent conditions” refers to conditionswhich allow formation of a so-called specific hybrid but do not allowformation of a non-specific hybrid (see, for example, Sambrook, J. etal., Molecular Cloning A Laboratory Manual, second Edition, Cold SpringHarbor Laboratory Press (1989)). Specific examples of the stringentconditions include performing hybridization in a solution containing 50%formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10× Denhardt's solution, and 100μg/mL salmon sperm DNA at 42° C., and washing at room temperature with2×SSC and a 0.1% SDS solution and at 50° C. with 0.1×SSC and a 0.1% SDSsolution.

Through introduction of such a DNA into virus, virus 1 of the presentinvention can be produced.

No particular limitation is imposed on the species of the virus intowhich such a DNA is introduced, so long as the virus is available fortransfection. The virus is preferably baculovirus. The baculovirus ispreferably NPV. No particular limitation is imposed on the species ofthe NPV, so long as the NPV is a virus belonging to NPVs. For example,AcNPV or Bombyx mori NPV (BmNPV) may be employed. Of these, AcNPV ispreferred.

Introduction of a DNA into virus may be performed through homologousrecombination by use of a transfer vector. No particular limitation isimposed on the type of the transfer vector. For example, pPSC8 (ProteinScience), pFastBac (Invitrogen), or pVL1393 (Pharmingen) may beemployed. Of these, pPSC8 is preferred. These transfer vectors may becommercial products.

No particular limitation is imposed on the method of homologousrecombination by use of a transfer vector. A specific example thereofwill be described later in the Examples.

Whether or not the produced virus harbors the aforementioned DNA (A) orDNA (B) may be confirmed by any of the following procedures: checkingthat the produced virus harbors a DNA encoding subunit α oflimulus-derived factor G through nucleotide sequence analysis; checkingthat a protein expressed by the produced virus has an amino acidsequence of subunit α of limulus-derived factor G; and checking that aprotein expressed by the produced virus has activity of subunit α oflimulus-derived factor G.

Virus 1 of the present invention may be used in the production of “thecell of the present invention” described later, and in “the method ofthe present invention.”

<1>-2 Virus 2 of the Present Invention

Virus 2 of the present invention is a virus harboring a DNA encodingsubunit β of limulus-derived factor G.

Examples of the horseshoe crab and preferred embodiments are the same asdescribed in <1>-1.

Particularly, the DNA harbored by virus 2 of the present invention ispreferably the following DNA (A) or (B):

(A) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 4,

(B) a DNA encoding a protein having an amino acid sequence defined bySEQ ID NO: 4 in which one or more amino acid residues are deleted,substituted, inserted, or transposed and having activity of subunit β oflimulus-derived factor G.

The DNA encoding a protein having an amino acid sequence defined by SEQID NO: 4 herein is a DNA encoding subunit β of factor G derived fromTachypleus tridentatus.

The definition of the protein encoded by DNA (B) is the same asdescribed in <1>-1. The protein encoded by DNA (B) has activity ofsubunit β of limulus-derived factor G. Since subunit β of factor G hasserine protease activity, subunit β activity can be confirmed by thepresence of serine protease activity.

Notably, the term “one or more amino acid residues” and the state“harboring a DNA” are the same as described in <1>-1. The method ofproducing virus 2 of the present invention is identical to thatdescribed in <1>-1, except that SEQ ID NO: 2 is changed to SEQ ID NO: 4.

The DNA (A) serving as a DNA encoding a protein having an amino acidsequence defined by SEQ ID NO: 4 may be, among others, a DNA having anucleotide sequence defined by nucleotides 1 to 930 in SEQ ID NO: 3.Alternatively, a DNA deposited in GenBank with an accession No. D16623may also be employed.

The aforementioned DNA (B) encoding a protein having an amino acidsequence defined by SEQ ID NO: 4 in which one or more amino acidresidues are deleted, substituted, inserted, or transposed and havingactivity of subunit β of limulus-derived factor G is the same asdescribed in <1>-1.

Through introduction of such a DNA into virus, virus 2 of the presentinvention can be produced. Examples of the virus into which the DNA isintroduced, preferred embodiments, and the DNA introduction method arethe same as described in <1>-1.

Whether or not the produced virus harbors the aforementioned DNA (A) orDNA (B) may be confirmed through the same method as described in <1>-1.

Virus 2 of the present invention may be used in the production of “thecell of the present invention” described later, and in “the method ofthe present invention.”

<2> The Cell of the Present Invention

The cell of the present invention harbors the virus of the presentinvention.

The virus of the present invention is the same as mentioned above.

No particular limitation is imposed on the cell to be employed, so longas the cell allows infection with the virus of the present invention,and can express, by the mediation of the virus of the present invention,subunits α and/or β of limulus-derived factor G. Examples of the cellinclude cells derived from insects, and specific examples include an Sf9cell.

No particular limitation is imposed on the method for causing the virusof the present invention to harbor the cell. For example, contactbetween the virus of the present invention and the cell readily causesinfection of the cell with the virus of the present invention, wherebythe cell can harbor the virus of the present invention. A specificmethod thereof will be described later in the Examples.

The cell of present invention may harbor sole virus 1 of the presentinvention, sole virus 2 of the present invention, or both viruses 1 and2 of the present invention. The cell may further harbor a virus otherthan viruses 1 and 2.

In the case where the cell of the present invention harbors viruses 1and 2 of the present invention, the cell is preferably produced byinfecting with the viruses 1 and 2 such that MOI of virus 1 exceeds MOIof virus 2. For example, the cell of the present invention may beinfected with viruses 1 and 2 at a ratio of MOI of virus 1 to MOI ofvirus 2 of 1.5:1 to 64:1. The ratio of MOI of virus 1 to MOI of virus 2is more preferably controlled to 1.5:1 to 32:1, 2:1 to 32:1, 2:1 to16:1, 2:1 to 8:1, 2:1 to 6:1, 2:1 to 4:1 or 3:1 to 5:1, 4:1, in thisorder.

Since the cell of the present invention can produce subunits α and/or βof limulus-derived factor G, the cell of the present invention may beselected on the basis of the production performance as an index.

The cell of the present invention may be employed in, for example, thebelow-mentioned method of the present invention.

<3> The Method of the Present Invention

The method of the present invention for producing subunit α and/orsubunit β of limulus-derived factor G includes growing the cell of thepresent invention and collecting subunit α and/or subunit β oflimulus-derived factor G from the growth product.

The cell of the present invention is the same as mentioned above.

In the present invention, the term “grow” refers to a concept includingproliferation of cells which are transformants and growing organismssuch as animals and insects into which transformant cells have beenincorporated. The term “growth product” is a concept including a culturemedium (supernatant of the culture) after completion of growth oftransformants, cultured cells themselves, and matter secreted orexcreted from organisms such as animals and insects into which the cellshave been incorporated.

No particular limitation is imposed on the growth conditions (e.g.,medium and culture conditions), so long as the cell of the presentinvention can grow and produce subunit α and/or subunit β oflimulus-derived factor G. The conditions are appropriately selected inaccordance with the type of the vectors, cells, etc. employed. Forexample, culturing temperature may be about 20 to 40° C.

The growth period of the cell of the present invention may also beappropriately tuned in accordance with the amount of the cell used inthe present invention, a desired production amount of the subunit(s),and other growth conditions.

The person skilled in the art may select the method for collectingsubunit α and/or subunit β of limulus-derived factor G from the growthproduct from generally employed methods in accordance with the type ofthe growth product.

For example, in the case where these subunits are produced in thesoluble form which are secreted into a culture medium (culturesupernatant), the culture medium is collected and may be employedwithout performing further treatment. In the case where these subunitsare produced in the soluble form which are secreted in the cytoplasm, orproduced in the insoluble form (membrane-binding), these subunits may beextracted through extraction with cell crushing such as the nitrogencavitation apparatus method, homogenizing, glass beads milling,sonication treatment, the permeation shock method, or freeze-thawing;extraction with a surfactant; or a combination thereof. The extractitself may be used as subunit α and/or subunit β without performingfurther treatment.

The method of the present invention may further include other steps, solong as the method includes growing the cell of the present inventionand collecting subunit α and/or subunit β of limulus-derived factor Gfrom the growth product. For example, the method may include a step ofpurifying the collected subunit(s). The purification may be incomplete(partial) purification or complete purification, and may beappropriately selected in accordance with the use purpose of thesubunit(s).

Specific examples of the purification method include salting out by themediation of a salt such as ammonium sulfate or sodium sulfate;centrifugation; dialysis; ultrafiltration; chromatographic methods suchas adsorption chromatography, ion-exchange chromatography, hydrophobicchromatography, reverse-phase chromatography, gel filtration, gelpermeation chromatography, and affinity chromatography; electrophoresis;and combinations thereof.

The method of the present invention may be employed for producing solesubunit α, sole subunit β, or both subunits α and β. The method may alsoproduce a subunit other than subunits α and β.

In the production of subunit α, a cell harboring virus 1 of the presentinvention is employed. In the production of subunit β, a cell harboringvirus 2 of the present invention is employed. In the production ofsubunits α and β, a cell harboring both viruses 1 and 2 of the presentinvention is employed.

In the case where both subunits α and β are produced, a protein which isformed of subunits α and β and which maintains activity oflimulus-derived factor G can be produced.

Whether or not the produced protein is subunit α and/or subunit β, isformed of subunits α and β, or maintains activity of limulus-derivedfactor G may be confirmed through analysis of the collected protein suchas amino acid sequence, molecular weight, electrophoresis features,Western blotting employing an antibody reacting specifically to therelevant subunit, BG binding performance, or presence of serine proteaseactivity.

The method of the present invention realizes remarkably effectiveproduction of a protein which is formed of subunit α, subunit β, orsubunits α and β and which maintains activity of limulus-derived factorG.

The method of the present invention includes a concept of “a method ofproducing factor G, the method comprising growing a cell which harbors aDNA encoding subunit α of factor G derived from a horseshoe crab and aDNA encoding subunit β of factor G derived from a horseshoe crab, andcollecting, from the growth product, a protein having activity of factorG derived from a horseshoe crab”.

EXAMPLES

The present invention will next be described in detail by way ofexamples.

<1> Expression of Subunit α of Factor G

A cDNA encoding factor G subunit α was kindly offered by Dr. TatsushiMUTA (Department of Molecular and Cellular Biochemistry, Graduate Schoolof Medical Sciences, Kyushu University). The cDNA had been preparedthrough a method disclosed in J. Biol. Chem., 269(2), p. 1370-1374(1994). The cDNA was introduced into a transfer vector (pPSC8), and aclone having a predetermined nucleotide sequence was selected. Thethus-selected expression vector (Factor G-α/pPSC8) DNA and a baculovirus(AcNPV) DNA were co-transfected into Sf9 cells. The virus fluid obtainedfrom the culture supernatant was purified and amplified. The viral DNAwas extracted from the cells infected with the baculovirus, andsequenced. Cells (expresSF+, trade name) were infected with thethus-obtained virus fluid, and the expression product was analyzedthrough Western blotting. Details of these steps will next be described.

1. Construction of Expression Vector

A cDNA encoding factor G subunit α (Factor G-α/pFastbac1) was treatedwith BamHI/Hind III, and fragments (about 2,100 bp) having a target genewere collected. The sample was blunt-ended, and subsequently, ligatedthrough mixing with Nru I-treated pPSC8 (product of Protein Science). E.coli JM109 was transformed with the ligation product, to thereby form atransformant. Plasmids in which fragments of the target size had beendetermined were purified, and sequenced. The sequencing was performed byuse of the below-described primers and ABI Prism Big Dye TerminatorCycle Sequencing Kit Ver.3 (Applied Biosystems). Electrophoresis wasperformed by means of an automated sequencer ABI Prism 310 GeneticAnalyzer (Applied Biosystems), and analysis was performed by means ofGenetyx (Genetyx). Sequences of the primers are shown in the followingsequence list by SEQ ID NOs: 5 to 13.

SEQ ID NO: 5: PSC F

SEQ ID NO: 6: PSC R

SEQ ID NO: 7: Factor G α 441/460-F

SEQ ID NO: 8: Factor G α 941/960-F

SEQ ID NO: 9: Factor G α 1601/1620-F

SEQ ID NO: 10: Factor G α 582/563-R

SEQ ID NO: 11: Factor G α 1082/1063-R

SEQ ID NO: 12: Factor G α 1582/1563-R

SEQ ID NO: 13: Factor G α 1700/1681-R

A clone in which insertion of a target gene had been confirmed wasinoculated to an LB medium (100 mL) containing 50 μg/mL ampicillin, andcultivated at 30° C. for one night. Proliferated cells were collected,and plasmids were purified in accordance with the manual of Plasmid MidiKit (QIAGEN).

2. Co-transfection

To Sf9 cells (1.0×106) plated in a 25-cm2 flask was added a serum-freeSF-900 II medium (product of Invitrogen) (200 μL) containing anexpression vector harboring a cDNA encoding factor G subunit α (4.6 μg),a linear AcNPV DNA (85 ng), and LIPOFECTIN Reagent (product ofInvitrogen) (5 μL). After the culture had been allowed to stand at 28°C. for six hours, a serum-free SF-900 II medium was further added so asto adjust the volume of the culture liquid to 5 mL. The culture wasfurther cultivated at 28° C. for nine days, and the culture supernatantwas collected. The thus-obtained solution through co-transfectionreferred to as a co-transfection solution.

3. Purification of Recombinant Virus

The recombinant virus was purified through the plaque assay method. Thespecific procedure is as follows.

Sf9 cells (2.0×106) were plated onto a plate (diameter: 60 mm) andallowed to stand at 28° C. for one hour, whereby the cells were adheredto the bottom surface. The aforementioned co-transfection solution wasdiluted with a serum-free Sf-900 II medium at dilution factors of 104,105, 106, and 107. An aliquot (1 mL) of each of these diluted solutionswas added to the cells, followed by gentle shaking at room temperaturefor one hour. After removal of the plate supernatant (virus fluid), aserum-free Sf-900 II medium (4 mL) containing 0.5% SeaKemGTG agarose(product of BMA) was added to the plate, and stationary culture wasperformed at 28° C. for seven days. From each culture medium, sixplaques of infected insect cells including no polyhedra were collected.The plaques of each medium were suspended in a serum-free Sf-900 IImedium (1 mL), to thereby serve as a virus fluid.

4. Amplification of Recombinant Virus

Next, amplification of the recombinant virus (preparation of recombinantvirus fluid) was performed. The specific procedure is as follows.

To Sf9 cells (2.0×106) plated in a 25-cm2 flask was added each (0.5 mL)of the aforementioned virus fluids, followed by stationary cultivationat 28° C. for one hour. A serum-free SF-900 II medium was added to theculture so as to adjust the volume of the culture liquid to 5 mL, andthe culture was further stationary-cultivated for three days, to therebyyield a first-generation virus fluid.

To Sf9 cells (6.0×106) plated in a 75-cm2 flask was added the entiretyof the aforementioned first-generation virus fluid, followed bystationary cultivation at 28° C. for one hour. Subsequently, aserum-free SF-900 II medium (10 mL) was added to the culture, followedby stationary cultivation for four days. After completion ofcultivation, cells were scraped out from the bottom of the flask by useof a cell scraper. The thus-collected cells were centrifuged at 3,000×gand 4° C. for 15 minutes, to thereby fractionate into the supernatantand the precipitate. The culture supernatant was collected and employedas a second-generation virus fluid.

5. Confirmation of Gene Insertion

Subsequently, insertion of a DNA into a cell was confirmed through thefollowing procedure.

The precipitate obtained at the collection of the second-generationvirus fluid was suspended in TE (200 μL), and a viral DNA was extractedin accordance with a manual of QIAamp DNA Mini Kit (QIAGEN). PCR wasperformed by use of the thus-extracted viral DNA as a template and thefollowing primers.

SEQ ID NO: 14: PSC F2

SEQ ID NO: 15: PSC R2

To a 0.2-mL sample tube, the aforementioned viral DNA (1 μL), 2.5 mMdNTP (8 μL), KOD buffer (5 μL), 25 mM magnesium chloride solution (4μL), primers PSC F2 and PSC R2 (4 pmol/mL each, 2.5 μL each), KOD DNApolymerase (product of TOYOBO) (1 μL), and sterilized pure water (26 μL)were added, and the mixture was sufficiently stirred. The mixture wassubjected to PCR for 30 cycles, each cycle consisting of 94° C. for 30seconds, 50° C. for 30 seconds, and 74° C. for 60 seconds.

The PCR product (5 μL) was subjected to electrophoresis on agarose gel,and the length of the amplified fragments was determined. A PCR productof a fragment having a target length was purified, and the sequences ofthe N-terminus side and the C-terminus side were determined, through useof the same reagents, apparatuses, and primers PSC F and PSC R asemployed in the aforementioned “1. Construction of expression vector.”

6. Production of Recombinant Virus Fluid

Insect cells (expresSF+, trade name, Protein Science) which were in thelogarithmic growth phase during cultivation were diluted with aserum-free Sf-900 II medium so as to adjust the concentration to 1.5×106cells/mL, and the diluted product (100 mL) was placed in a 250-mLErlenmeyer flask. The aforementioned second-generation virus fluid (1mL) was added thereto, and the mixture was subjected to shakecultivation at 130 rpm and 28° C. for three days. After completion ofcultivation, the culture liquid was centrifuged at 3,000×g and 4° C. for15 minutes, to thereby fractionate into the supernatant and theprecipitate. The culture supernatant was collected and employed as athird-generation virus fluid.

7. Titer Determination

Sf9 cells (2.0×106) were plated onto a plate (diameter: 60 mm) andallowed to stand at 28° C. for one hour, whereby the cells were adheredto the bottom surface. Subsequently, the culture liquid was removed.Separately, the third-generation virus fluid was diluted with aserum-free Sf-900 II medium at dilution factors of 105, 106, 107, and108. An aliquot (1 mL) of each of these solutions was added to theplate, followed by gentle shaking at room temperature for one hour.After removal of the plate supernatant (virus fluid), a serum-freeSf-900 II medium (4 mL) containing 0.5% SeaKemGTG agarose (product ofBMA) was added to the plate, and stationary culture was performed at 28°C. for nine days. In each culture medium, the number of observed plaqueswas counted, thereby determining the titer.

8. Expression Test

Insect cells (expresSF+) were diluted with a serum-free Sf-900 II mediumso as to adjust the concentration to 1.5×106 cells/mL, and the dilutedproduct (100 mL/per flask) was placed in three 250-mL Erlenmeyer flasks.The aforementioned third-generation virus fluid was added thereto so asto attain MOIs of 0.5, 2, and 8, respectively. Each mixture wassubjected to shake cultivation at 130 rpm and 28° C. for three days.After completion of cultivation, the culture liquid was centrifuged at3,000×g and 4° C. for 15 minutes, to thereby fractionate into thesupernatant and the precipitate.

9. Detection of Expression Product

Each of the samples collected in “8. Expression test” above wassubjected to SDS-PAGE through a routine method. A protein wastransferred to a blotting membrane through the semi-dry blotting method,and the expression product was detected by Western blotting under thebelow-mentioned conditions. Note that the DNA encoding factor G subunitα incorporated into the virus had been designed so as to express aHis-tag-bound protein. Sample treatment: The supernatant was mixed withLaemmli Sample Buffer (product of BIO-RAD), and the mixture was heatedat 99° C. for three minutes. The precipitate (200 μL) was mixed with PBS(200 μL), to thereby form a suspension. Laemmli Sample Buffer was addedto the suspension, and the mixture was heated at 99° C. for threeminutes.

-   Amount of applied sample: 20 μL/lane-   SDS-PAGE gel: 12.5% gel (product of BIO-RAD)-   Voltage application in SDS-PAGE: 150V, CV-   Blotting membrane: PVDF-   Voltage application in blotting: 15V, CV, 30 minutes-   Antibody: Penta His HRP Conjugate (product of QIAGEN)-   Detection: ECL Detection Reagent (product of Amersham Biosciences)    10. Results

Analysis of the total nucleotide sequence after insertion to pPSC8indicates that the obtained nucleotide sequence completely coincideswith that of the DNA encoding factor G subunit α. Therefore, no mutationwas found to be introduced through PCR. The nucleotide sequence analysisof the N-terminal portion and C-terminal portion of the target sequencein the recombinant virus has revealed that the nucleotide sequences ofthe two portions completely coincide with those of the DNA encodingfactor G subunit α. Thus, the recombinant virus was found to have anucleotide sequence of the DNA encoding factor G subunit α.

The titer was determined to be 3×108 pfu/mL.

In the results of “9. Detection of expressed product” above, a bandattributed to reaction with an anti-His-Tag antibody was observed at atarget position (about 75 kDa). Thus, expression of factor G subunit αwas confirmed.

<2> Expression of Subunit β of Factor G

A cDNA encoding factor G subunit β was kindly offered by Dr. TatsushiMUTA (Department of Molecular and Cellular Biochemistry, Graduate Schoolof Medical Sciences, Kyushu University). The cDNA had been as preparedthrough a method disclosed in J. Biol. Chem., 269(2), p. 1370-1374(1994). Factor G subunit β was expressed through the same procedure asemployed in <1> above, and the expression product was analyzed. Detailsof these steps will next be described.

1. Construction of Expression Vector

A cDNA encoding factor G subunit β (Factor G-β/pFastbac1) was treatedwith BamHI/Hind III, and fragments (about 1,000 bp) having a target genewere collected. The sample was blunt-ended, and subsequently, ligatedthrough mixing with Nru I-treated pPSC8. E. coli JM109 was transformedwith the ligation product, to thereby form a transformant. A clone inwhich insertion of a target gene had been confirmed was inoculated to anLB medium (100 mL) containing 50 μg/mL ampicillin, and cultivated at 37°C. for one night. Proliferated cells were collected, and plasmids werepurified in accordance with the manual of Plasmid Midi Kit (QIAGEN).

2. Co-transfection

To Sf9 cells (1.0×106) plated in a 25-cm2 flask was added a serum-freeSF-900 II medium (200 μL) containing an expression vector harboring acDNA encoding factor G subunit β (4.6 μg), a linear AcNPV DNA (85 ng),and LIPOFECTIN Reagent (5 μL). After the culture had been allowed tostand at 28° C. for six hours, a serum-free SF-900 II medium was furtheradded so as to adjust the volume of the culture liquid to 5 mL. Theculture was further cultivated at 28° C. for seven days, and the culturesupernatant was collected, to thereby serve as a co-transfectionsolution.

3. Purification of Recombinant Virus

The recombinant virus was purified through the plaque assay method. Thespecific procedure is as follows.

Sf9 cells (2.0×106) were plated onto a plate (diameter: 60 mm) andallowed to stand at 28° C. for one hour, whereby the cells were adheredto the bottom surface. The aforementioned co-transfection solution wasdiluted with a serum-free Sf-900 II medium at dilution factors of 104,105, 106, and 107. An aliquot (1 mL) of each of these solutions wasadded to the cells, followed by gentle shaking at room temperature forone hour. After removal of the plate supernatant (virus fluid), aserum-free Sf-900 II medium (4 mL) containing 0.5% SeaKemGTG agarose(product of BMA) was added to the plate, and stationary culture wasperformed at 28° C. for six days. From each culture medium, six plaquesof infected insect cells including no polyhedra were collected. Theplaques of each medium were suspended in a serum-free Sf-900 II medium(1 mL), to thereby serve as a virus fluid.

4. Amplification of Recombinant Virus

Next, amplification of the recombinant virus (preparation of recombinantvirus fluid) was performed. The specific procedure was as follows.

To Sf9 cells (2.0×106) plated in a 25-cm2 flask was added each (0.5 mL)of the aforementioned virus fluids, followed by stationary cultivationat 28° C. for one hour. A serum-free SF-900 II medium was added to theculture so as to adjust the volume of the culture liquid to 5 mL, andthe culture was further stationary-cultivated for three days, to therebyyield a first-generation virus fluid.

To Sf9 cells (6.0×106) plated in a 75-cm2 flask was added the entiretyof the aforementioned first-generation virus fluid, followed bystationary cultivation at 28° C. for one hour. Subsequently, aserum-free SF-900 II medium (10 mL) was added to the culture, followedby stationary cultivation for four days. After completion ofcultivation, cells were scraped out from the bottom of the flask by useof a cell scraper. The thus-collected cells were centrifuged at 3,000×gand 4° C. for 15 minutes, to thereby fractionate into the supernatantand the precipitate. The culture supernatant was collected and employedas a second-generation virus fluid.

5. Confirmation of Gene Insertion

Subsequently, insertion of a DNA into a cell was confirmed through thefollowing procedure.

The precipitate obtained at the collection of the second-generationvirus fluid was suspended in TE (200 μL), and a viral DNA was extractedin accordance with a manual of QIAamp DNA Mini Kit (QIAGEN). PCR wasperformed by use of the thus-extracted viral DNA as a template and thefollowing primers.

SEQ ID NO: 16: PSC F2

SEQ ID NO: 17: PSC R2

To a 0.2-mL sample tube, the aforementioned viral DNA (1 μL), 2.5 mMdNTP (8 μL), KOD buffer (5 μL), 25 mM magnesium chloride solution (4μL), primers PSC F2 and PSC R2 (4 pmol/mL each, 2.5 μL each), KOD DNApolymerase (product of TOYOBO) (1 μL), and sterilized pure water (26 μL)were added, and the mixture was sufficiently stirred. The mixture wassubjected to PCR for 30 cycles, each cycle consisting of 94° C. for 30seconds, 50° C. for 30 seconds, and 74° C. for 60 seconds.

The PCR product (5 μL) was subjected to electrophoresis on agarose gel,and the length of amplified fragments was determined. A PCR product of afragment having a target length was purified, and the sequences of theN-terminus side and the C-terminus side were determined, through use ofthe same reagents and apparatuses as employed in the aforementioned“<1>-1. Construction of expression vector.” The following primers wereemployed.

SEQ ID NO: 18: PSC F

SEQ ID NO: 19: PSC R

6. Production of Recombinant Virus Fluid

Insect cells (expresSF+, trade name, Protein Science) which were in thelogarithmic growth phase during cultivation were diluted with aserum-free Sf-900 II medium so as to adjust the concentration to 1.5×106cells/mL, and the diluted product (100 mL) was placed in a 250-mLErlenmeyer flask. The aforementioned second-generation virus fluid (1mL) was added thereto, and the mixture was subjected to shakecultivation at 130 rpm and 28° C. for three days. After completion ofcultivation, the culture liquid was centrifuged at 3,000×g and 4° C. for15 minutes, to thereby fractionate into the supernatant and theprecipitate. The culture supernatant was collected and employed as athird-generation virus fluid.

7. Titer Determination

Sf9 cells (2.0×106) were plated onto a plate (diameter: 60 mm) andallowed to stand at 28° C. for one hour, whereby the cells were adheredto the bottom surface. Subsequently, the culture liquid was removed.Separately, the third-generation virus fluid was diluted with aserum-free Sf-900 II medium at dilution factors of 105, 106, 107, and108. An aliquot (1 mL) of each of these solutions was added to theplate, followed by gentle shaking at room temperature for one hour.After removal of the plate supernatant (virus fluid), a serum-freeSf-900 II medium (4 mL) containing 0.5% SeaKemGTG agarose (product ofBMA) was added to the plate, and stationary culture was performed at 28°C. for nine days. In each culture medium, the number of observed plaqueswas counted, thereby determining the titer.

8. Expression Test

Insect cells (expresSF+) were diluted with a serum-free Sf-900 II mediumso as to adjust the concentration to 1.5×106 cells/mL, and the dilutedproduct (100 mL/per flask) was placed in three 250-mL Erlenmeyer flasks.The aforementioned third-generation virus fluid was added thereto so asto attain MOIs of 0.5, 2, and 8, respectively. Each mixture wassubjected to shake cultivation at 130 rpm and 28° C. for three days.After completion of cultivation, the culture liquid was centrifuged at3,000×g and 4° C. for 15 minutes, to thereby fractionate into thesupernatant and the precipitate.

9. Detection of Expression Product

Each of the samples collected in “8. Expression test” above wassubjected to SDS-PAGE and Western blotting through the same method asemployed in “<1>-9. Detection of expression product. Note that the DNAencoding factor G subunit β incorporated into the virus had beendesigned so as to express a His-tag-bound protein.

10. Results

The nucleotide sequence analysis of the N-terminal portion andC-terminal portion of the target sequence in the recombinant virus hasrevealed that the nucleotide sequences of the two portions completelycoincide with those of the DNA encoding factor G subunit β. Thus, therecombinant virus was found to have a nucleotide sequence of the DNAencoding factor G subunit β.

The titer was determined to be 1.7×108 pfu/mL.

In the results of “9. Detection of expressed product” above, a bandattributed to reaction with an anti-His-Tag antibody was observed at atarget position (about 37 kDa). Thus, expression of factor G subunit βwas confirmed.

<3> Co-Expression of Subunits α and β of Factor G

The third-generation virus fluids prepared in <1> and <2> above forproducing factor G subunits α and β, respectively, were employed so asto co-express both subunits.

Insect cells (expresSF+) were diluted with a serum-free Sf-900 II mediumso as to adjust the concentration to 1.5×106 cells/mL, and the dilutedproduct (50 mL/per flask) was placed in three 125-mL Erlenmeyer flasks.The aforementioned third-generation virus fluids, which had beenprepared for producing factor G subunits α and β, were added thereto atthe following proportions. Each mixture was subjected to shakecultivation at 130 rpm and 28° C. for three days. After completion ofcultivation, the culture liquid was centrifuged at 3,000×g and 4° C. for15 minutes, to thereby fractionate into the supernatant and theprecipitate. The supernatant was frozen for preservation.

Sample 1:

subunit α:subunit β=1:0 (by MOI)

subunit α:subunit β=57.7:0 (by virus amount (μL))

Sample 2:

subunit α:subunit β=0:1 (by MOI)

subunit α:subunit β=0:187.5 (by virus amount (μL))

Sample 3:

subunit α:subunit β=1:1 (by MOI)

subunit α:subunit β=57.7:187.5 (by virus amount (μL))

Sample 4:

subunit α:subunit β=1:2 (by MOI)

subunit α:subunit β=57.7:375 (by virus amount (μL))

Sample 5:

subunit α:subunit β=1:4 (by MOI)

subunit α:subunit β=57.7:750 (by virus amount (μL))

Sample 6:

subunit α:subunit β=2:1 (by MOI)

subunit α:subunit β=115.4:187.5 (by virus amount (μL))

Sample 7:

subunit α:subunit β=4:1 (by MOI)

subunit α:subunit β=230.8:187.5 (by virus amount (μL))

The procedure as employed in <1>-9 above was repeated, except that 10%gel (product of BIO-RAD) was employed as the SDS-PAGE gel and ananti-GST-HRP Conjugate (product of Amersham Biosciences) was employed asan antibody for detection, to thereby perform SDS-PAGE and Westernblotting of the supernatants. Note that the DNA encoding factor Gsubunit α and that encoding factor G subunit β incorporated into thevirus had been designed so as to express GST-bound proteins.

As a result, bands attributed to reaction with an anti-GST antibody wereobserved at target positions (about 75 kDa and 37 kDa). Thus, expressionof factor G subunits α and β was confirmed.

Separately, the supernatant was purified by using Ni Sepharose 6 FastFlow (product of Amersham Biosciences). After desalting andconcentration of the eluate, the procedure as employed in <1>-9 abovewas repeated, except that 5-20% gradient gel (product of ATTO) wasemployed as the SDS-PAGE gel, mixture of an anti-Factor G subunit αserum and an anti-Factor G subunit β serum (kindly offered by Dr.Tatsushi MUTA (Department of Molecular and Cellular Biochemistry,Graduate School of Medical Sciences, Kyushu University)) was employed asa first antibody, HRP-conjugated anti-rabbit IgG antibody was employedas a second antibody and Konica Immunostain HRP-100 (product of KonicaMinolta) was employed as a reagent for detection, to thereby performSDS-PAGE and Western blotting of the eluate. Note that the DNA encodingfactor G subunit α and that encoding factor G subunit β incorporatedinto the virus had been designed so as to express His-tag-boundproteins.

As a result, bands attributed to reaction with the mixture of ananti-Factor G subunit α serum and an anti-Factor G subunit β serum wereobserved at target positions (about 75 kDa and 37 kDa). Thus, expressionof factor G subunits α and β was confirmed.

After cultivation, the supernatant was collected from each of theaforementioned seven samples, and whether or not the expressed proteinmaintains factor G activity was checked.

Specifically, the supernatant fraction after completion of cultivationwas diluted at a factor of 11 times with an ice-cooled 50 mM Tris-HClbuffer (pH: 7.5) containing 150 mM NaCl. To the diluted product (25 μL),there were added a pro-clotting enzyme derived from a lysate (25 μL),dextran (final concentration: 2.4%), Tris-HCl buffer (pH: 8.0) (finalconcentration: 0.08 M), MgSO4 (final concentration: 0.08 M), CaCl2(final concentration: 0.16 mM), distilled water for injection (10 μL),Boc-Leu-Gly-Arg-pNA substrate (see Japanese Patent Application laid-Open(kokai) No. 08-122334) (final concentration: 0.53 mM), and BG (0.25 ng),followed by adjusting the total volume to 125.1 μL. The mixture wasallowed to react at 37° C. for 24 hours. After completion of reaction,absorbance of the sample was measured at 405 nm (blank) and 492 nm.Factor G derived from a lysate was employed as a positive control. Theexperiment was performed twice, and absorbance measures were averaged.The results are as follows.

Results:

Sample 1 (α:β=1:0): 0.166

Sample 2 (α:β=0:1): 0.167

Sample 3 (α:β=1:1): 0.278

Sample 4 (α:β=1:2): 0.190

Sample 5 (α:β=1:4): 0.169

Sample 6 (α:β=2:1): 0.730

Sample 7 (α:β=4:1): 1.078

factor G: 1.328

As is clear from the results, Samples 6 and 7, which had been preparedwith controlling a MOI of virus harboring a DNA encoding subunit α to behigher than a MOI of virus harboring a DNA encoding subunit β duringinfection of cells with the virus, were found to have factor G activity.The analysis also indicated that intrinsic functions of thethus-expressed subunits α and β were not impaired.

The thus-produced factor G can be reacted with BG. Therefore,as-produced factor G may be employed for assaying BG or diagnosingmycosis.

As described hereinabove, the present invention provides a tool and amethod for mass-expressing factor G derived from a horseshoe crab or asubunit forming the factor. Factor G and subunit(s) forming the factorproduced according to the present invention may be employed asassay/diagnosis tools for BG assay, mycosis diagnosis, and otherpurposes, as well as laboratory reagents.

1. A method of producing recombinant factor G derived from horseshoecrab, the method comprising growing an insect-derived cell, which hasbeen co-transfected with a first baculovirus harboring Tachvpleustridentatus cDNA encoding wild-type subunit α of factor G and a secondbaculovirus harboring Tachvpleus tridentatus cDNA encoding wild-typesubunit β of factor G, such that the multiplicity of infection in saidinsect cell for the first baculovirus is higher than the secondbaculovirus, and wherein the α subunit and β subunit come together toform active recombinant factor G.
 2. The method according to claim 1,wherein the DNA encoding wild-type subunit α of factor G is DNA encodingthe polypeptide defined by SEQ ID NO: 2 and the DNA encoding wild-typesubunit β of factor G is DNA encoding the polypeptide defined by SEQ IDNO:
 4. 3. The method according to claim 1, wherein the DNA encodingwild-type subunit α of factor G is DNA having the nucleotide sequencedefined by nucleotides 1 to 2022 in SEQ ID NO: 1 and the DNA encodingwild-type subunit 13 of factor G is DNA having the nucleotide sequencedefined by nucleotides 1 to 930 in SEQ ID NO:
 3. 4. The method accordingto claim 1, wherein the baculovirus is nuclear polyhedrosis virus. 5.The method according to claim 1, wherein the ratio of multiplicity ofinfection of the first baculovirus to the second baculovirus is from1.5:1 to 64:1.